An endogenous growth hormone secretagogue (GHS) for a growth hormone secretagogue receptor (GHS-R) which is one of orphan receptors was purified and isolated from rat stomach in 1999, and was named ghrelin (Kojima et al., Nature, vol. 402, p. 656–660, 1999). This peptide is known to have a characteristic structure in which a hydroxy group of a serine residue is acylated with a fatty acid. Further, ghrelin in which a serine residue or a threonine residue at the 3-position contains a fatty acid-modified site was isolated also from a vertebrate other than rat, such as human, mouse, pig, fowl, eel, cow, horse, sheep, frog, trout or dog, or was presumed from a cDNA (Table 1). For example, human ghrelin consists of 28 amino acids, and the serine side chain at the 3-position is acylated with a fatty acid (N-octanoic acid). It has been found that this novel peptide has strong growth hormone secretagogue activity, and modification of 3-positional serine or threonine with a fatty acid is essential for manifestation of the activity (Kojima et al., Nature, vol. 402, p. 656–660, 1999). In addition, it has been clarified that ghrelin secreted from stomach functions as a blood hormone in regulating secretion of growth hormone, and thus much attention has been paid to a physiological role of ghrelin and its application to medicaments.
TABLE 1HumanGSS (n-octanoyl) FLSPEHQRVQQRKESKKPPAKLQPRGSS (n-octanoyl) FLSPEHQRVQRKESKKPPAKLQPR RatGSS (n-octanoyl) FLSPEHQKAQQRKESKKPPAKLQPRGSS (n-octanoyl) FLSPEHQKAQRKESKKPPAKLQPR MouseGSS (n-octanoyl) FLSPEHQKAQQRKESKKPPAKLQPR PorcineGSS (n-octanoyl) FLSPEHQKVQQRKESKKPAAKLKPR BovineGSS (n-octanoyl) FLSPEHQKLQRKEAKKPSGRLKPR OvineGSS (n-octanoyl) FLSPEHQKLQRKEPKKPSGRLKPR CanineGSS (n-octanoyl) FLSPEHQKLQQRKESKKPPAKLQPR EelGSS (n-octanoyl) FLSPSQRPQGKDKKPPRV-NH2 TroutGSS (n-octanoyl) FLSPSQKPQVRQGKGKPPRV-NH2GSS (n-octanoyl) FLSPSQKPQGKGKPPRV-NH2 ChickenGSS (n-octanoyl) FLSPTYKNIQQQKGTRKPTARGSS (n-octanoyl) FLSPTYKNIQQQKDTRKPTARGSS (n-octanoyl) GLSPTYKNIQQQKDTRKPTARLH BullfrogGLT (n-octanoyl) FLSPADMQKIAERQSQNKLRHGNMGLT (n-decanoyl) FLSPADMQKIAERQSQNKLRHGNMGLT (n-octanoyl) FLSPADMQKIAERQSQNKLRHGNMN TilapiaGSS (n-octanoyl) FLSPSQKPQNKVKSSRI-NH2 CatfishGSS (n-octanoyl) FLSPTQKPQNRGDRKPPRV-NH2GSS (n-octanoyl) FLSPTQKPQNRGDRKPPRVG EquineGSS (n-butanoyl) FLSPEHHKVQHRKESKKPPAKLKPR
In addition to an octanolyl group-(C8)-modified peptide, there are butanoyl group (C4)-, hexanoyl group (C6)-, decanoyl group (C10)- and dodecanoyl group (C12)-modified peptides. Furthermore, there are unsaturated fatty acid-modified peptides.
Some peptides or proteins, like ghrelin and cholecystokinin, manifest their physiological role when a specific amino acid residue in the amino acid sequence has undergone modification such as acylation, sulfonation, glycosylation or phosphorylation. It is thought that these modifications are given by an elaborate enzyme system in a living body, and a general method for producing a modified peptide or protein in a high quality and effective manner in large quantities has not been reported yet. For example, since ghrelin exhibits growth hormone secretagogue activity by modification of a specific amino acid side chain with a long chain fatty acid, fatty acid modification is an essential structural element. However, what enzyme system of a living body is involved for ester-binding of a fatty acid to a hydroxy group of a specific amino acid side chain or for extention of a fatty acid has not been known yet. Since, in particular, ghrelin is the first physiologically active peptide clarified to have a structure in which a hydroxy group of an amino acid side chain is modified with a fatty acid, although the present peptide is a useful peptidic hormone which is expected to be a promising medicament as a curative medicine for eating disorder, a drug for promoting growth hormone, secretion, etc., production of a peptide having fatty acid modification in a specific hydroxy-containing amino acid side chain has not been generalized. That is, an industrial production method which is advantageous for mass production of such peptide has not been established at present.
Currently, various peptide or protein preparations such as insulin, growth hormone, calcitonin, atrial natriuretic peptide, LH-RH derivative and adrenocorticotropic hormone derivative are used as a medicament. As a method for producing these peptides or proteins, production by a chemical synthesis method, an enzymatic method and a genetic recombination method are known. Although, which method to be employed is appropriately selected, generally a chemical synthesis method is selected when the number of residues is small, and an enzymatic method or a genetic recombination method is selected when the number of residues is large.
A chemical synthesis method, for example, is a method by which a physiologically active peptide or protein having modification such as ghrelin, etc. can be steadily produced. Many production methods using a chemical synthesis method have been already reported as a method for producing a modified peptide or protein. In the case of ghrelin, methods are reported by Bednarek et al. (J. Med. Chem., vol. 43, p. 4370–4376, 2000) and Matsumoto et al. (Biochem. Biophys. Res. Commun., vol. 284, p. 655–659, 2001). Also, International Publication No. WO 01/07475 describes a production method by a chemical synthesis method, as a method for producing a peptide which is ghrelin or a ghrelin derivative, or a salt thereof. However, in a production by a chemical synthesis method, there is usually a limitation to a chain length of a peptide which can be synthesized, while retaining constant quality (purity). Although a liquid phase chemical synthesis method can synthesize a peptide of high purity, the method is not common for synthesis of a long chain peptide due to the solubility, long producing step and special techniques necessary for the reaction treatment. Namely, effective production in large quantities is difficult in a production method using a liquid phase chemical synthesis method. On the other hand, a solid phase chemical synthesis for extending a peptide chain on a resin has a simplified step, and is more advantageous for mass production, but this method also has a limitation on a constructable chain length to obtain desired products having constant quality. In addition, there is also a problem that the method is inferior in economic property because of excessive reagents used, in particular, in production of a long chain peptide.
Meanwhile, a method for enzymatically coupling a peptide fragment such as an enzymatic method is excellent in that protection of an amino acid side chain can be minimized. In this method, however, since a reverse reaction of hydrolysis is usually used, the condition setting is in principle difficult, and thus the method is not practical.
On the other hand, production of a physiologically active peptide or protein by a genetic recombination method is a useful production method suitable for mass production. However, in a method using a prokaryote such as Escherichia coli having high productivity, it is difficult to directly produce a peptide having a modified site, since a prokaryote has no posttranslational modification system. In a genetic recombination method using a eukaryote such as yeast and various egg cells, modification such as glycosylation, acylation, sulfonation and phospholyration is possible, however, regarding a fatty acid, for example, it is difficult to introduce only fatty acid having a constant length. Among isolated ghrelins, ghrelins having not only octanoic acid (C8) but also butanoic acid (C4), decanoic acid (C10), or unsaturated fatty acid of them have been found, and thus it is clear that controlling the introduction of a fatty acid having a specific chain length is difficult. In addition, since productivity by a eukaryote is generally low, production system of yeast and various egg cells having modification system has much room for improvement from a viewpoint of mass production of a modified peptide or protein such as ghrelin.
As described above, although a chemical synthesis method has been known as a method for synthesizing a modified peptide or protein, and there are already various reports, such method has a room for improvement in yield and cost for the purpose of mass production. It is difficult to directly produce a modified peptide or protein by a genetic recombination method using a prokaryote such Escherichia coli. Also, production by a genetic recombination method using a eukaryote such as yeast has a problem in unity or productivity, and thus there is a room for improvement in order to overcome this problem. In an enzymatic method using a reverse reaction of hydrolysis, it is difficult to set respective condensation conditions and, therefore, this method cannot be said an advantageous method for mass production.
As described above, there has been a room for further improvement in an effective production of a peptide or a protein having modification such as glycosylation, acylation, sulfonation, phosphorylation, etc., which satisfies quality and quantitative elements, by the independent application of a conventionally known chemical synthesis method, an enzymatic method or a genetic recombination method alone.
Then, as one of methods utilizing advantages of the aforementioned methods and compensating the defects of the methods, there may be exemplified a semi-synthesis method obtained by combination of a chemical synthesis method and a genetic recombination method. An important point of the production method is to effectively produce a peptide fragment in a form suitable for condensation. A peptide fragment having a modified amino acid residue (hereinafter, also referred to as modified component) and a peptide fragment having no modified amino acid residue (hereinafter, also referred to as non-modified component) may be in an N-terminal side or a C-terminal side, or there may be a plurality of modified components. A production method can be designed appropriately depending on a desired peptide or protein. As one example, the case where a modified component is present on an N-terminal side, and another peptide fragment to be condensed with the peptide fragment (modified component) is a non-modified component will be described in detail.
A native chemical ligation method (Dawson et al., Science, vol. 266, p. 776–779, 1994) which has been paid attention to recently has a defect that a Cys residue remains on a ligation site. Recently, however, a thioester method obtained by improvement of the aforementioned method has been proposed. For example, Kawakami et al. have reported a phosphorylated peptide synthesis by a method using thioester (Tetrahedron Letter, vol. 41, p. 2625–2628, 2000).
A specific example of the thioester method will be described below. In an example of synthesis of a phosphorylated p21Max protein (Kawakami et al., Tetrahedron Lett., vol. 39, p. 7901–7904, 1998), a peptide fragment (modified component)(13 mer) containing a phosphoric acid-modified site is produced as thioester by a solid phase chemical synthesis method. On the other hand, a peptide fragment having a sequence in which one amino acid residue is added to the N-terminus of a non-modified component is produced in Escherichia coli, glyoxylic acid is acted on this in the presence of a divalent copper or nickel ion to convert an amino acid residue added to the N-terminus into an α-ketoacyl group, and a side chain amino group is protected with a Boc group. Then, an α-ketoacyl group is removed with phenylenediamine, thereby a peptide fragment (non-modified component) in which only an amino acid group in the N-terminal amino acid residue is freed is produced. Finally, these both fragments are condensed by adding an active esterifying agent such as silver salt, excessive HOOBt, etc.
Also in the aforementioned method, there still remains the following problem. There is a problem on stability of the thioester in production of a peptide fragment (modified component), and it is reported that the yield is 11%. In addition, for production of a peptide fragment (non-modified component), the said method using an α-ketoacyl group is a potential choice as a chemical method of freeing a N-terminal amino group. However, the method has a safety problem in that an α-ketoacyl group is unstable, and a mutagenic substance such as phenylenediamine is used for eliminating the group, when a physiologically active peptide or protein for a medicament is produced. In addition, an active esterifying agent such as silver salt, excessive HOOBt, etc. is used in a reaction of condensing both fragments and, therefore, there is also a problem on racemization, toxicity and cost.
A semi-synthesis method combining a chemical synthesis method and a genetic recombinant method is described in International Publication No. WO 01/07475 as a method for producing a peptide which is ghrelin or a ghrelin derivative, or a salt thereof. More specifically, there is described a method for producing rat ghrelin (1–28) by condensing rat ghrelin (1–5) prepared by a chemical synthesis method with rat ghrelin (6–28) prepared by a genetic recombination method. However, since such method also has the following problem, there is a room for much improvement in order to obtain an efficient and industrially advantageous production method. That is, there is a room for improving productivity in consideration of that, when rat ghrelin (1–5) is obtained by eliminating a peptide chain with TFA from a resin, a Boc group and a t-Bu group are at the same time removed, and thus it is necessary to introduce again a Boc group into a N-terminus, and that since a Ser side chain becomes unprotected, a strong activating agent can not be used for condensation with rat ghrelin (6–28), etc. Furthermore, in a process of condensing protected rat ghrelin (1–5) and protected rat ghrelin (6–28), a C-terminal amino acid of an acylpeptide fragment part may be racemized, and therefore there is a room for improvement in order to prevent this problem. Herein, ghrelin (m–n) means a peptide having an amino acid sequence of mth to nth from an N-terminus of ghrelin. Hereinafter, the same.
In addition, there are some problems in preparation of a protected peptide fragment (non-modified component). A method for producing protected rat ghrelin (6–28) described in International Publication No. WO 01/07475 fundamentally adopts a two-step enzyme treatment method (International Publication NO. WO 99/38984) and, as a processing enzyme therefor, two kinds of enzymes, i.e. a recombinant V8 protease derivative (rV8D5)(JP-A No. 9-47291) and a Kex2 protease (JP-A No. 10-229884), are used. However, since a plasmid (pG97s rGR) expressing a fusion protein containing protected rat ghrelin (6–28) is constructed based on a plasmid (JP-A No. 9-296000) which highly expresses a fusion protein of Escherichia coli β-galactosidase derivative and human parathyroid hormone (1–34), a linker sequence suitable for its amino acid sequence may need to be selected in preparation of a protected peptide fragment (non-modified component).
Furthermore, since there is phenomenon that a protecting group for protected rat ghrelin (6–28) is eliminated in an aqueous solution, a recovery rate in purification step is very low as 10%, and therefore there is still a problem for stable supply of ghrelin in large quantities.
As described above, even a combination of a chemical synthesis method and a genetic recombinant method has a further problem in order to realize a method for efficiently producing a modified physiologically active peptide or protein, which is very safe enough to be used as a medicament.